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Technology

Large-scale Genotyping

The Affymetrix GeneChip® Human Mapping 10K Array is a Single Nucleotide Polymorphism (SNP) genotyping tool based on allele-specific hybridization. By synthesizing probes on the array corresponding to both of the two possible alleles at each SNP and hybridizing the target to the array, it can be determined whether a SNP is AA, AB, or BB by analyzing the resulting signal from the allele-specific probes.

Forty different 25mer oligonucleotides are tiled for each SNP represented, each oligonucleotide with a slight variation in perfect matches, mismatches, and flanking sequence around the SNP (illustration of principle).

The assay requires 250 nanograms of genomic DNA, which is digested with a restriction enzyme (XbaI), resulting in fragments of various sizes (simplified graphical overview). Adapters are ligated to all fragments regardless of their size, but fragments in the 250- to 1,000-bp size range are preferentially amplified by PCR using a generic primer that recognizes the adapter sequence. The amplified DNA is then fragmented, end-labeled and hybridized to the GeneChip® Mapping 10K Array. After scanning the array Affymetrix Software (GDAS 2.0) is used to calculate a call for each individual genotype. With 11.555 SNP’s represented on the array and a call rate of greater than 90% more than 10.000 genotypes should be expected per sample.



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